1. Field of the Invention
This invention relates to an isolated DNA comprising a gene and a method of using the DNA. More specifically, this invention relates to a gene encoding a protein inhibiting the action of human microphthalmia-associated transcription factor (MITF) and the expression of the gene, and a method for evaluating the ability of human melanocytes to express the above protein, using a DNA fragment from the gene.
2. Description of the Related Art
The microphthalmia gene (mi) of mice is known, based on their mutant strains lacking the gene, to be involved in phenotypes such as the loss of pigmentation, small eyes, the defection of mast cells and abnormality in bones. This gene is located on chromosome No. 6. Since the absence of the mi gene results in abnormalities in different areas, it is suggested that the gene is involved in various controls in the embryological stage.
This mouse mi gene was recently cloned. As a result of analyses of the gene, it was found that the gene encodes a transcription factor protein having a basic-helix-loop-helix-zipper structure (hereinafter referred to as xe2x80x9cb-HLH-ZIP structurexe2x80x9d) which is known to be involved in protein-protein interactions (Hodgkinson, C. A. et al., Cell, 74, 395-404 (1993); Hughes, M. J. et al., J. Biol. Chem., 268, 20687-20690 (1993)). Thereafter, a human mi gene (hereinafter referred to as xe2x80x9cmitfxe2x80x9d) which is homologous to the mouse mi gene, was also cloned, and was found to be located on chromosome No. 3 (Tachibana, M. et al., Hum. Mol. Genet., 3, 553-557 (1994); Yasumoto, K. et al., Mol. Cell. Biol., 14, 8058-8070 (1994)).
Incidentally, it is known that the cell-specific expression of a tyrosinase gene is controlled by several positive and negative elements existing in the promoter region. It was suggested that the expression of the gene of human tyrosinase is controlled by four positive elements and one negative element, and when the MITF as the product of a human mitf gene is bound to two E-box motifs (CATGTG) contained on the promoter region of the tyrosinase gene, the promoter activity of the tyrosinase gene is heightened (Bentley, N. J., et al., Mol. Cell. Biol., 14, 7996-8006 (1994)). Likewise, it is also reported that in mice, when the Mi as the product of a mouse mi gene is bound to a M-box (a motif conserved by the promoters of tyrosinase, TRP-1 and TRP-2) as one of the positive elements, the promoter activity of the tyrosinase gene is heightened (Ganss, R., et al., J. Biol. Chem., 269, 29808-29816 (1994)).
Further, it has been clarified that mouse Mi protein forms a family as a homodimer or a heterodimer with TFEB, TFE3 or TFEC (each thereof is b-HLH-ZIPmi) and binds to DNAs (Hemesath, T. J. et al., Gene and Dev., 8, 2770-2780 (1994)).
Under these findings, the present inventors began searching for proteins which interact with human MITF as a target molecule. The inventors believed that if proteins which can interact with human MITF and act inhibitorily thereon, could be identified, each of the molecules could be expected to inhibit melanin formation in mammal cells at an initial stage. Such substances involved in the melanin formation system have a possibility, for example, to serve as agents for controlling whitening or tanning in the cosmetic and dermatological field from the viewpoint of, so-called, pigment cell biology.
Heretofore, as substances having whitening action, there have been, for example, mentioned tyrosinase inhibitors, endothelin inhibitors, reducing agents of melanin intermediate products, etc. However, these substances either inhibit partial melanin synthesis stimulation input, or, under some circumstances, reduce or inhibit the production of melanin which are continuously formed at the site of melanin deposition, etc. Therefore, these substances are not sufficient since they do not stop the causes of melanin deposition, etc. As a result, compounding substances having whitening action which control basic melanin synthesis are sought to overcome the problems of prior art substances.
Namely, if a protein interacting with human MITF as a target molecule and a gene encoding it exist, and the protein can inhibit the action of human MITF, they will serve as a control of the basic pigment synthesis system.
The present inventors used a two hybrid system in the identification of a protein which interacts with human MITF as a target molecule. As a result, they succeeded in obtaining a protein which specifically binds to human MITF but whose functions are unclear. Further, this protein was the same as the Cys2/His2 zinc finger protein (rKr2 for xe2x80x9crat kruppel-type proteinxe2x80x9d) derived from rat oligodendrocytes (U. Pott et al., J. Neurochem., Vol.65, No.5, 1995, 1955-1966).
When the functions of this rKr2 protein were examined, it was found that rKr2 binds to the human MITF protein via its basic domain, and thereby inhibits MITF protein binding to the promoter regions of tyrosinase and TRP1. In other words, by this process, rKr2 acts to inhibit the activation of the tyrosinase and TRP1 genes. Further, when rat rKr2 was actually introduced into human melanocytes, the expression of TRP1 was inhibited. It was thus, concluded from these results that rat rKr2 has an action to inhibit melanin synthesis.
Based on this result, the present inventors expected that a gene homologous to a rat rKr2-like gene also existed in human melanocytes, and have vigorously researched for such a gene. As a result, they finally succeeded in obtaining a gene homologous to the rat rKr2 gene, namely a cDNA encoding a protein binding to human MITF, from a human cDNA library. Thus, they succeeded first in confirming the presence of a rKr2 gene in a human being (hereinafter, also referred to as human rKr2 gene), and found that this human rKr2 gene also has melanin synthesis-inhibiting effect in a human melanocyte-culturing system.
Therefore, according to this invention, there is provided an isolated DNA comprising a gene encoding a protein which binds to and inhibits the action of a human MITF. As specific embodiments of the DNA, there can be mentioned:
1) An isolated DNA comprising a gene which is at least 85% homologous to a rat rKr2 gene and corresponds to base numbers 1843-2109 of the rat rKr2 gene,
2) An isolated DNA comprising a nucleotide sequence which hybridizes to the gene of (1) above or its complement under stringent conditions, and encodes a protein which binds to and inhibits the action of human MITF, but is not the same as the rat rKr2 gene, or
3) An isolated DNA comprising a human gene having a nucleotide sequence as set forth in SEQ ID NO:1.
When the DNA according to the present invention is, for example, integrated into a suitable expression vector and the resultant vector is introduced into melanocytes of a mammal, especially a human being, to express the DNA, the production of melanin in the melanocytes can be controlled. Therefore, the DNA may, for example, be used for the treatment of diseases caused by abnormal melanin accumulation.
Further, the invention also provides proteins such as those containing a human rKr2 protein, which can be obtained by the expression of the DNA according to the present invention. Such proteins may be used as a tool for researches into the elucidation of the mechanism of the melanin production system in human melanocytes, or as an effective ingredient for whitening cosmetics.
As another embodiment of the invention, there is provided a method for evaluating the ability of a human melanocyte to express a protein which binds to and inhibits the action of a human MITF, using a primer prepared based on the above DNAs, particularly a DNA comprising a human gene homologous to a rat rKr2 gene or a nucleotide sequence hybridizing to the human gene above or its complement under stringent conditions and encoding a protein binding to human MITF, to inhibit the action of human MITF. According to the evaluation method, an ability of human melanocytes, particularly an ability relating to melanin production, can be evaluated. Such evaluation results may not only make the classification of melanocytes possible, but also, for example, when the results show an abnormal value, make it possible to provide a judgment criterion for taking appropriate treatment.
As another embodiment of the invention, there is provided a method for screening a substance having an influence on the expression of a protein which binds to and inhibits the action of a human MITF, in human melanocytes which comprises evaluating and screening whether or not certain substances have such an influence, using a primer prepared based on the above DNAs, particularly a DNA comprising a human gene homologous to a rat rKr2 gene, or a nucleotide sequence encoding a protein binding to human MITF, to inhibit the action of human MITF.
According to the screening method, when the pigment synthesis system of human melanocytes can be influenced by a substance so as to increase the expression of the protein, the substance can be screened as a substance inhibiting melanin deposition in human melanocytes, and, conversely, when the substance can have an influence so as to decrease the expression of the protein, the substance can be screened as a substance accelerating melanin deposition in human melanocytes. Since it can be thought that the thus screened substance can basically or primitively act on the melanin synthesis system in human melanocytes, substances of a category different from those of conventional whitening agents or tanning agents can, for example, be provided.